携有胰岛素样生长因子1重组腺病毒对链脲佐菌素诱导胰岛β细胞损害的保护性研究

目的构建含有鼠胰岛素样生长因子-1(rIGF-1)重组腺病毒,研究rIGF-1在链脲佐菌素(STZ)诱导胰岛β细胞损害中的作用。方法构建重组腺病毒并感染RINm5F细胞,ELISA、Western blotting检测rIGF-1蛋白表达;STZ诱导细胞破坏,检测培养上清中NO水平,胰岛素释放试验进行功能测定,流式细胞检测细胞凋亡,四甲基偶氮唑盐法检测细胞存活率。结果成功构建含有rIGF-1基因重组腺病毒,病毒滴度为4.0×108pfu/ml,rIGF-1在细胞内外有效表达,并具有抑制STZ诱导胰岛β细胞凋亡和NO产生、保护细胞胰岛素分泌和促进细胞增殖的活性。结论胰岛β细胞局部表达IGF-1能够保护细胞功能,抑制NO产生,使胰岛β细胞免受诱导凋亡因子的损害,提高细胞存活率。STZ诱导胰岛β细胞凋亡可能与NO介导有关。

Protective effect of recombinant adenovirus carrying rat insulin-like growth factor 1 on rat islet β-cell against strepozotocin-induced impairment in vitro

OBJECTIVE: To construct the recombinant adenovirus containing rat insulin-like growth factor 1 (rIGF-1), and then infect to rat islet beta cells-RINm5F cells with the virus to investigate the role of rIGF-1 to streptozotocin-induced cell impairment in vitro. METHODS: Recombinant adenovirus encoding rIGF-1 was constructed, and then infect to RINm5F cells. rIGF-1 protein was detected by Western blot analysis and ELISA method. Then streptozotocin was used to induce RINm5F cells impairment. The levels of nitric oxide were detected in cells culture supernatants. The cells function was evaluated by glucose-stimulated insulin production. The apoptosis was analyzed by flow cytometry. Thiaoollyl blue viability assay was applied to exam the number of viable cells. RESULTS: The recombined adenovirus-rIGF-1 was constructed successfully and its titer was about 4.0x10(8) pfu/ml. The rIGF-1 was expressed in the RINm5F cells and cell culture supernatants. rIGF-1 expression could inhibit islet cells apoptosis, significantly decrease the level of NO induced by streptozotocin and significantly increase insulin secretion and cells viability. CONCLUSION: These results suggested that the high concentration of rIGF-1 in cultured islets may be beneficial in maintaining islet beta cells function and protecting islet beta cells from apoptosis-mediated factors. The apoptosis induced by STZ may be NO-dependent.